The current tuberculosis vaccine, BCG, has questionable efficacy and undoubtedly new vaccines are needed. Recent studies have indicated that DNA vaccines may be promising as new mycobacterial vaccines candidates. We have generated 30 DNA vaccines expressing different mycobacterial antigens. All of these vaccines have been tested by intramuscular inoculation,either singly or in combination, for their capacity to induce protective, cytokine, and humoral immune responses in mice. Based on the results of these studies, we have concluded the following: 1. At least 10 tuberculosis DNA vaccines that we have tested induce protective immune responses in mice. However, the level of protection for single vaccines is less than the level of protection evoked by BCG. 2. Tuberculosis DNA vaccines encoding mycobacterial antigens fused to a TPA signal signal generally are more immunogenic than DNA vaccines encoding the native protein. TB DNA vaccines encoding mycobacterial antigens fused to ubiquitin induce primarily a class I response with high levels of cell- mediated immunity and low humoral responses generated. 3. Combinations of TB DNA vaccines do evoke a larger protective response than single vaccines. The level of protective immunity elicited by some combination TB DNA vaccines is similar to the BCG response. In a long-term survival study, mice immunized with a combination of ten TB DNA vaccines survived 15-fold longer than naive mice after a high-dose aerogenic challenge with virulent M. tuberculosis. We currently testing other combinations of TB DNA vaccines in long term studies to establish whether these preparations can also induce extended protective immunity. To further define the specific immune responses responsible for protective immunity to tuberculosis, we have evaluated the susceptibility to TB infection of mice deficient in the CIITA gene. The CIITA gene is the IFN-regulated master transactivator of MHC class II expression. In low dose aerogenic challenge studies, we have shown that CIITA knockout mice are highly susceptible to tuberculosis. The median time to death for these KO mice is about 60 days while wild-type mice survive about 300 days after a low dose challenge. FACS analyses of splenocytes from KO mice indicate that only CD8 T cells are induced after TB infection of these mice and not the CD4 T cells that are largely responsible for controlling TB infections. As a result of their failure to generate immune CD4 cells, IFN-gamma levels in the KO mice (in response to the infection) are dramatically reduced relative to wild-type mice. This study demonstrates that the MHC class II pathway is an essential component of the protective immune response to TB and that new vaccines must be designed to stimulate class II immunity.